Oligonucleotide compounds have important therapeutic applications in medicine. For example, certain oligonucleotides can be used to silence genes that are associated with particular diseases. Gene-silencing prevents formation of a protein by inhibiting translation. Importantly, gene-silencing agents represent a promising alternative to traditional small, organic compounds that inhibit the function of a protein linked to a disease.
dsRNA directs the sequence-specific degradation of mRNA through a process known as RNA interference (RNAi). RNAi is an evolutionarily conserved gene silencing mechanism, originally discovered in studies of the nematode Caenorhabditis elegans (Lee et al, Cell 75:843 (1993); Reinhart et al., Nature 403:901 (2000)). It is triggered by introducing dsRNA into cells expressing the appropriate molecular machinery, which then degrades the corresponding endogenous mRNA. The mechanism of RNAi involves conversion of dsRNA into short RNAs that direct ribonucleases to homologous mRNA targets (summarized, Ruvkun, Science 2294:797 (2001)). This process is related to normal defense mechanisms against viruses and the mobilization of transposons, and occurs in a wide variety of organisms, including mammals and other vertebrates.
The use of recombinantly produced dsRNA molecules or chemically synthesized oligonucleotides of the same or similar nature thus enables the targeting of specific mRNAs for silencing in mammalian cells. For example, small interfering RNA (siRNA) molecules are dsRNA molecules that exploit the RNAi mechanism within cells by targeting specific mRNAs of interest for degradation. As such, these dsRNA molecules represent promising agents for a variety of diagnostic and therapeutic purposes. For example, siRNA compounds can be used to identify the function of genes. In addition, siRNA compounds offer enormous potential as a new type of pharmaceutical agent which acts by silencing disease-causing genes. siRNA therapeutic agents for the treatment of many diseases including central-nervous-system diseases, inflammatory diseases, metabolic disorders, oncology, infectious diseases, and ocular disease, are under active development.
Despite the recent advances in siRNA technology, the need exists for siRNA molecules having improved pharmacologic properties. Requirements for successful implementation of siRNA therapeutic molecules, which are generally designed to target disease-causing genes to prevent the production of their encoded proteins, include (a) stability in vivo, (b) sufficient membrane permeability and cellular uptake, and (c) a good balance of binding affinity and sequence specificity. Many oligonucleotide analogs have been developed in which the phosphodiester linkages of native DNA are replaced by other linkages that are more resistant to nuclease degradation (see e.g. Barawkar and Bruice 1998; Linkletter, Szabo et al. 2001; Micklefield 2001). In addition, oligonucleotides have been modified by conjugation with other molecules, including peptides, in order to enhance cellular uptake (e.g., Moulton, Nelson et al. 2004; Nelson, Stein et al. 2005).
Nevertheless, there remains a need for modified dsRNA structures with improved performance, particularly in the area of stability and specificity, without compromising sequence selectivity. The present invention satisfies this need and offers other related advantages.